Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm

We report on two-photon fluorescence excitation (TPE) action cross sections for five widely used molecular fluorophores. Measurements were performed by use of ultrashort (~100-fs) Ti:sapphire pulsed excitation over the range 690-960 nm. TPE spectra were obtained by comparison with a fluorescein calibration standard. Large cross sections were found for the cyanine reagent Cy 3 (~140 GM) and for Rhodamine 6G (~150 GM), both at 700 nm [1 GM = 10(-50) (cm(4) s)/photon]. Several fluorophores show interesting and desirable blue shifts with respect to twice the one-photon absorption wavelength. Fluorophore fluorescence intensities showed no significant departure (?4%) from quadratic illumination power dependence, indicating genuine two-photon processes. Implications of these measurements for two-photon laser-scanning microscopy are discussed.